My goal in obtaining this K99 award is to acquire the additional training to become an independent investigator in the field of single-molecule studies of telomere protein-DNA and protein-protein interactions. This study will be under the guidance of Dr. Van Houten and sponsored by Drs. Erie and Opresko. The expertise and resources from these three labs provide an excellent environment for me to advance my skills in AFM and single-molecule fluorescence imaging, telomere protein biochemistry, and QPCR assays. We hypothesize that, in addition to disrupting TRF1, TRF2 and POT1 proteins binding to DMA, environmentally-induced DMA damage (such as UV light or oxidative stress) at telomeres can cause stochastically unstable assemblies of telomere binding proteins on DMA. This in turn progressively favors the disruption of the T-loop structure and the exposure of the 3' overhang. The specific aims of this study are two-fold. The first aim is to evaluate the effects of environmentally-induced DMA damage on G-quadruplex formation, protein binding, protein assemblies, and T-loop formation. We will use AFM to examine the effects of DMA damage on G-quadruplex assembly. The impact of bulky DMA lesions on the binding of POT1, TRF1 and TRF2 to short telomeric DMA substrates will be evaluated using electrophoresis mobility shift assays (EMSAs), and the T-loop formation will be examined using AFM. In AFM studies, loading of POT1 (marked by quantum dots, QDs) onto duplex telomeric DMA will be used as a reporter of shelterin assembly. The second aim is to evaluate the effects of DNA damage on dynamics of protein-DNA interaction and protein assembly on telomeric DNA. We will characterize the functionality of TRF1, TRF2, and POT1-QD conjugates using AFM imaging and EMSA. These protein-QD conjugates will be used in single-molecule fluorescence studies to evaluate how UV-induced DNA damage affects the dynamics of TRF1-, TRF2-DNA interactions and shelterin assembly. It is known that certain environmental DNA damaging agents cause increased telomere shortening. Short telomeres are characteristic of various human diseases. This study will greatly advance our understanding of how exposure to environmental stressors. such as UV light and oxidative stress, is associated with telomere dysfunction in the etiology of several human disorders including age-associated degenerative diseases and cancer.